This application claim priority under 35 U.S.C. 119 of Danish application 1332/97 filed Nov. 21, 1997, the contents of which are fully incorporated herein by reference.
This invention relates to novel mutant protease enzymes or enzyme variants, comprising insertions in one or more active site loops, useful in formulating detergent compositions and exhibiting improved wash performance in detergents; cleaning and detergent compositions containing said enzymes; mutated genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and such host cells transformed therewith and capable of expressing said enzyme variants.
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM(copyright) (Novo Nordisk A/S), RELASE(copyright) (Novo Nordisk A/S), MAXAPEM(copyright) (Gist-Brocades N.V.), PURAFECT(copyright) (Genencor International, Inc.).
Further a number of protease variants are described in the art, such as in EP 130756 (GENENTECH)(corresponding to US Reissue Patent No. 34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050 (GENEX); EP 260105 (GENENCOR); Thomas, Russell, and Fersht (1985) Nature 318 375-376; Thomas, Russell, and Fersht (1987) J. Mol. Biol. 193 803-813; Russel and Fersht Nature 328 496-500 (1987); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95/27049.(SOLVAY S.A.); WO 95/30011 (PROCTER and GAMBLE COMPANY); WO 95/30010 (PROCTER and GAMBLE COMPANY); WO 95/29979 (PROCTER and GAMBLE COMPANY); US 5.543.302 (SOLVAY S.A.); EP 251 446 (GENENCOR); WO 89/06279 (NOVO NORDISK A/S); WO 91/00345 (NOVO NORDISK A/S); EP 525 610 Al (SOLVAY); and WO 94/02618 (GIST-BROCADES N.V.).
However, even though a number of useful protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses.
Therefore, an object of the present invention, is to provide improved proteases or protein engineered protease variants, especially for use in the detergent industry.
The present inventors have identified that it is possible to construct variants of BLSAVI (Savinase(copyright)), having improved wash performance in detergent, as compared to the parent wildtype BLSAVI, by introducing at least one insertion in at least one of the active site loops in said BLSAVI.
It is predicted that it will be possible to make similar variants in other subtilases, which are similar to BLSAVI.
Further it is predicted that it is possible to isolate from nature and identify naturally occurring parent or wildtype subtilases, having improved wash performance in a detergent, as compared to BLSAVI, by specifically screening for such parent wildtype subtilases comprising at least one active site loop, which is longer than the corresponding active site loop in BLSAVI.
Accordingly, in a first aspect the invention relates to an isolated subtilase enzyme,having improved wash performance in a detergent, as compared to BLSAVI, having an amino acid sequence which is at least 40% identical to the amino acid sequence of the mature BLSAVI, and characterized by that at least one of the active site loops, in said isolated subtilase, is longer than the corresponding active site loop in BLSAVI, whereby such active site loops regions, in said isolated subtilase, is having the minimum amino acid length as specified from the group below comprising:
(a) the region (both of the end amino acids included) between amino acid residue from 33 to 43 is at least 11 amino acid long (i.e. at least one amino acid insertion, as compared to BLSAVI);
(b) the region (both of the end amino acids included) between amino acid residue 95 to 103 is at least 9 amino acids long (i.e. at least one amino acid insertion, as compated to BLSAVI);
(c) the region (both of the end amino acids included) between amino acid residue 125 to 132 is at least 8 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI);
(d) the region (both of the end amino acids included) between amino acid residue 153 to 173 is at least 21 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI);
(e) the region (both of the end amino acids included) between amino acid residue 181 to 195 is at least 15 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI);
(f) the region (both of the end amino acids included) between amino acid residue 202 to 204 is at least 3 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI); and
(g) the region (both of the end amino acids included) between amino acid residue 218 to 219 is at least 3 amino acids long (i.e. at least one amino acid insertion, as compared to BLSAVI).
In a second aspect the invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.
In a third aspect the invention relates to an expression vector comprising an isolated DNA sequence encoding a subtilase variant of the invention.
In a fourth aspect the invention relates to a microbial host cell transformed with an expression vector according to the fourth aspect.
In a further aspect the invention relates to the production of the subtilisin enzymes of the invention by inserting an expression vector according to the fourth aspect into a suitable microbial host, cultivating the host to express the desired subtilase enzyme, and recovering the enzyme product.
Further the invention relates to a composition comprising a subtilase variant of the invention.
Even further the invention relates to the use of the mutant enzymes for a number of industrial relevant uses, in particular for use in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes.
Definitions
Prior to discussing this invention in further detail, the following term will first be defined.
Nomenclature of Amino Acids
A=Ala=Alanine
V=Val=Valine
L=Leu=Leucine
I=Ile=Isoleucine
P=Pro=Proline
F=Phe=Phenylalanine
W=Trp=Tryptophan
M=Met=Methionine
G=Gly=Glycine
S=Ser=Serine
T=Thr=Threonine
C=Cys=Cysteine
Y=Tyr=Tyrosine
N=Asn=Asparagine
Q=Gln=Glutamine
D=Asp=Aspartic Acid
E=Glu=Glutamic Acid
K=Lys=Lysine
R=Arg=Arginine
H=His=Histidine
X=Xaa=Any amino acid
Nomenclature of Nucleic Acids
A=Adenine
G=Guanine
C=Cytosine
T=Thymine (only in DNA)
U=Uracil (only in RNA)
Nomenclature of Varianrs
In describing the various enzyme variants produced or contemplated according to the invention, the following nomenclatures have been adapted for ease of reference:
Original amino acid(s) position(s) substituted amino acid(s)
In the case when the original amino acid residue may be any amino acid residue, a short hand notation may at times be used indicating only the position and substituted amino acid,
Position Substituted Amino Acid
Such a notation is particular relevant in connection with modifications) in homologous subtilases (vide intra).
Similarly when the identity of the substituting amino acid residue(s) is immaterial,
Original Amino Acid Position
When both the original amino acid(s) and substituted amino acid(s) may comprise any amino acid, then only the position(s) is indicated, e.g.: 170.
When the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), then the selected amino acids are indicated inside brackets { }. Original amino acid position {substituted amino acid1, . . . , substituted amino acidn}
For specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue.
Substitutions
The substitution of Glutamic acid for glycine in position 195 is designated as:
Gly195Glu or G195E
or the substitution of any amino acid residue acid for glycine in position 195 is designated as:
Glu195Xaa or G195X
or
Glu195 or G195
The substitution of serine for any amino acid residue in position 170 would thus be designated
Xaa170Ser or X170S.
or
170Ser or 170S
Such a notation is particular relevant in connection with modification(s) in homologous subtilases (vide infra). 170Ser is thus meant to comprise e.g. both a Lys170Ser modification in BASBPN and Arg170Ser modification in BLSAVI. See FIG. 1 in relation to these examples.
For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of glycine, alanine, serine or threonine for arginine in position 170 would be indicated by
Arg170 {Gly,Ala,Ser,Thr}or R170{G,A,S,T} to indicate the variants
R170G, R170A, R170S, and R170T.
Deletions
A deletion of glycine in position 195 will be indicated by:
Gly195* or G195*
Correspondingly the deletion of more than one amino acid residue, such as the deletion of glycine and leucine in positions 195 and 196 will be designated
Gly195*+Leu196* or G195*+L196*
Insertions
The insertion of an additional amino acid residue such as e.g. a lysine after G195 is
Gly195GlyLys or G195GK; or
when more than one amino acid residue is inserted, such as e.g. a Lys, Ala and Ser after G195 this is
Gly195GlyLysAlaSer SEQ. ID NO: 1 or G195GKAS
In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example the sequences 194 to 196 would thus be:
194 195 196
BLSAVI A-G-L
194 195 195a 195b 195c 196
Variant A-G-K-A-S-L (SEQ. ID NO: 2)
In cases where an amino acid residue identical to the existing amino acid residue is inserted it is clear that a kind of degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G195GG. The same actual change could just as well be indicated as A194AG for the change from
194 195 196
BLSAVI A-G-L
to
194 195 195a 196
Variant A-G-G-L (SEQ. ID NO: 3)
194 194a 195 196
Such instances will be apparent to the skilled person, and the indication G195GG and corresponding indications for this type of insertions are thus meant to comprise such equivalent degenerate indications.
Filling a Gap p Where a deletion in an enzyme exists in comparison to the subtilisin sequence used for the numbering, an insertion in such a position is indicated as:
*36Asp or *36D
for the insertion of an aspartic acid in position 36
Multiple Modifications
Variants comprising multiple modifications are separated by pluses, e.g.:
Arg170Tyr+Gly195Glu or R170Y+G195E representing modifications in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.
or e.g. Tyr167{Gly,Ala,Ser,Thr}+Arg170 {Gly,Ala,Ser,Thr} designates the variants
Tyr167Gly+Arg170Gly, Tyr167Gly+Arg170Ala,
Tyr167Gly+Arg170Ser, Tyr167Gly+Arg170Thr,
Tyr167Ala+Arg170Gly, Tyr167Ala+Arg170Ala,
Tyr167Ala+Arg170Ser, Tyr167Ala+Arg170Thr,
Tyr167Ser+Arg170Gly, Tyr167Ser+Arg170Ala,
Tyr167Ser+Arg170Ser, Tyr167Ser+Arg170Thr,
Tyr167Thr+Arg170Gly, Tyr167Thr+Arg170Ala,
Tyr167Thr+Arg170Ser, and Tyr167Thr+Arg170Thr.
This nomenclature is particular relevant relating to modifications aimed at substituting, replacing, inserting or deleting amino acid residues having specific common properties, such as residues of positive charge (K, R, H), negative charge (D, E), or conservative amino acid modification(s) of e.g. Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr}, which signifies substituting a small amino acid for another small amino acid. See section xe2x80x9cDetailed description of the inventionxe2x80x9d for further details.
Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3).
Numbering of Amino Acid Positions/Residues
Unless otherwise stated the amino acid numbering used herein correspond to that of the subtilase BPNxe2x80x2 (BASBPN) sequence. For further description of the BPNxe2x80x2 sequence see Siezen et al., Protein Engng. 4 (1991) 719-737 and FIG. 1.
Serine Proteases
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 xe2x80x9cPrinciples of Biochemistry,xe2x80x9d Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
Subtilases
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases have been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. and FIG. 1 herein.
One subgroup of the subtilases, I-S1, comprises the xe2x80x9cclassicalxe2x80x9d subtilisins, such as subtilisin 168, subtilisin BPNxe2x80x2, subtilisin Carlsberg (ALCALASE(copyright), NOVO NORDISK A/S), and subtilisin DY.
A further subgroup of the subtilases I-S2, is recognized by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL(copyright), Gist-Brocades NV), subtilisin 309 (SAVINASE(copyright), NOVO NORDISK A/S), subtilisin 147 (ESPERASE(copyright), NOVO NORDISK A/S), and alkaline elastase YaB.
xe2x80x9cSavinase(copyright)xe2x80x9d
SAVINASE(copyright) is marketed by NOVO NORDISK A/S. It is subtilisin 309 from B. Lentus and differs from BABP92 only in one position (N87S, see FIG. 1 herein). SAVINASE(copyright) has the amino acid sequence designated BLSAVI (see FIG. 1 herein).
Parent Subtilase
The term xe2x80x9cparent subtilasexe2x80x9d is a subtilase defined according to Siezen et al. (Protein Engineering 4:719-737 (1991)). For further details see description of xe2x80x9cSUBTILASESxe2x80x9d immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modification have been made while retaining the characteristic of a subtilase.
Alternatively the term xe2x80x9cparent subtilasexe2x80x9d may be termed xe2x80x9cwild-type subtilasexe2x80x9d.
Modification(s) of a Subtilase Variant
The term xe2x80x9cmodification(s)xe2x80x9d used in connection with modification(s) of a subtilase variant as discussed herein is defined to include chemical modification as well as genetic manipulation. The modification(s) can be by substitution, deletion and/or insertions in or at the amino acid(s) of interest.
Subtilase Variant
In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.
Homologous Subtilase Sequences
Specific active site loop regions, and amino acid insertions in said loops of the subtilase SAVINASE(copyright) are identified for modification herein to obtain a subtilase variant of the invention.
However, the invention is not limited to modifications of this particular subtilase, but extend to other parent (wild-type) subtilases, which have a homologous primary structure to that of SAVINASE(copyright).
In order to identify other homologous subtilases, within the scope of this invention, an alignment of said subtilase(s) to a group of previously aligned subtilases is performed keeping the previous alignment constant. A comparison to 18 highly conserved residues in subtilases is performed. The 18 highly conserved residues are shown in table I (see Siezen et al. for further details relating to said conserved residues).
After aligning allowing for necessary insertions and deletions in order to maintain the alignment suitable homologous active site loop regions are identified. Said homologous residues can then be modified according to the invention.
Using the CLUSTALW (version 1.7, June 1997) computer alignment program (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) Nucleic Acids Research, 22:4673-4680.), using default alignment parameters, alignment of a given subtilase to a group of previously aligned subtilases is achieved using the Profile alignments option in the program. For a given subtilase to be within the scope of the invention, preferably 100% of the 18 highly conserved residues should be conserved. However, alignment of greater than or equal to 17 out of the 18 residues, or as little as 16 of said conserved residues is also adequate to identify homologous residues. Conservation of the, in subtilases, catalytic triad Asp32/His64/Ser221 should be maintained.
An alignment of 10 subtilases as defined is shown in FIG. 1.
Further in said process to identify a homologous parent (wild-type) subtilase within the scope of the invention, the 18 conserved residues above relates to the parent (wild-type) primary sequence of said homologous parent subtilase. In other words, if a parent subtilase has been modified in any of said 18 conserved residues above, it is the original parent wild-type sequence in said 18 conserved residues, which determines whether or not both the original parent subtilase and a possible variant of said parent subtilase, which is modified in any of said 18 conserved residues above, is a homologous subtilase within the scope of the present invention.
Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases and corresponding homologous active site loop regions, which can be modified according to the invention.
Wash Performance
The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, wash-ability, detergency, or wash performance. Throughout this application the term wash performance will be used to encompass this property.
Isolated DNA Sequence
The term xe2x80x9cisolatedxe2x80x9d, when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5xe2x80x2 and 3xe2x80x2 untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). The term xe2x80x9can isolated DNA sequencexe2x80x9d may alternatively be termed xe2x80x9ca cloned DNA sequencexe2x80x9d.
Isolated Protein
When applied to a protein, the term xe2x80x9cisolatedxe2x80x9d indicates that the protein is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. xe2x80x9chomologous impuritiesxe2x80x9d (see below)). An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE. Further it is preferred to provide the protein in a highly purified form, i.e., more than 40% pure, more than 60% pure, more than 80% pure, more preferably more than 95% pure, and even more preferably more than 99% pure, as determined by SDS-PAGE.
The term xe2x80x9cisolated proteinxe2x80x9d may alternatively be termed xe2x80x9cpurified proteinxe2x80x9d.
Homologous Impurties
The term xe2x80x9chomologous impuritiesxe2x80x9d means any impurity (e.g. another polypeptide than the polypeptide of the invention) which originate from the homologous cell where the polypeptide of the invention is originally obtained from.
Obtained from
The term xe2x80x9cobtained fromxe2x80x9d as used herein in connection with a specific microbial source, means that the polynucleotide and/or polypeptide produced by the specific source, or by a cell in which a gene from the source have been inserted.
Substrate
The term xe2x80x9cSubstratexe2x80x9d used in connection with a substrate for a protease should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a protease.
Product
The term xe2x80x9cproductxe2x80x9d used in connection with a product derived from a protease enzymatic reaction should in the context of this invention be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction.